DROPLET-BASED SELECTION

• US 20120015382A1
• Filed: 08/01/2011
• Published: 01/19/2012
• Est. Priority Date: 07/13/2007
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Weitz, David A., Griffiths, Andrew, Koester, Sarah, Mootha, Vamsi K., Duan, Honey, Agresti, Jeremy, Merten, Christoph, Heyman, John, Gilbert, John R.
• Application Number:
  US13/195,468
• Publication Number:
  US 20120015382A1
• Time in Patent Office: Days
• US Class Current: 435/7.92
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/149 Sorting the particles
• CPC Class Codes: G01N 33/505 involving T-cells
• CPC Class Codes: G01N 33/5052 involving B-cells
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

The present invention generally relates to fluidic droplets, and techniques for screening or sorting such fluidic droplets. In some embodiments, the fluidic droplets may contain cells (e.g., hybridoma cells) that can secrete various species, such as antibodies, for example. In one aspect, a plurality of fluidic droplets containing cells is screened to determine proteins, antibodies, polypeptides, peptides, nucleic acids, or the like. For example, cells able to secrete species such as antibodies may be selected according to certain embodiments of the invention. Examples of such cells include, for instance, immortal cells such as hybridomas, or non-immortal cells such as B-cells. For instance, blood cells may be encapsulated within a plurality of fluidic droplets, and the cells able to produce antibodies may be determined. In some cases, expression or secretion levels may be determined using signaling entities, for example, determinable microparticles present within the fluidic droplet. Other aspects of the invention relate to kits involving such fluidic droplets, methods of promoting the making or use of such fluidic droplets, and the like.

Claims

1-225. -225. (canceled)

226. A method of determining binding of a protein to a particle contained within a fluidic droplet, the method comprising acts of:

(a) providing a microfluidic channel containing a fluid containing microfluidic droplets having an average diameter of less than about 1 mm, wherein at least one microfluidic droplet contains;

(1) a protein-producing cell, and (2) a particle having immobilized thereon a protein-binding agent;

(b) causing the protein-producing cell within the microfluidic droplet to secrete, into the fluid, a protein;

(c) causing binding of the secreted protein in the fluid to the particle via the protein-binding agent; and

(d) determining at least one characteristic of the secreted protein by determining a characteristic of the particle contained in the droplet, after binding of the protein to the protein-binding agent immobilized on the particle.

227. The method of claim 226, further comprising sorting the microfluidic droplet based on the determined at least one characteristic of the protein.

228. The method of claim 226, wherein the protein-producing cell is a blood cell.

229. The method of claim 228, wherein the blood cell is removed from a subject.

230. The method of claim 226, wherein the protein-producing cells are encapsulated in the plurality of microfluidic droplets at an average ratio of no more than about 1 cell/microfluidic droplet.

231. The method of claim 226, wherein the protein is an antibody.

232. The method of claim 231, wherein the droplet further comprises a target to which the antibody is able to bind, and a signaling entity able to bind the target.

233. The method of claim 232, further comprising determining binding of the antibody to the binding agent and the target, thereby determining selectivity of binding of the antibody to the target.

234. The method of claim 226, wherein the microfluidic droplets are substantially monodisperse.

235. The method of claim 226, further comprising cloning DNA from the protein-producing cell.

236. The method of claim 235, wherein the DNA is amplified prior to cloning.

237. The method of claim 235, further comprising inserting at least a portion of the DNA in a host cell, and culturing the host cell to express the DNA.

238. The method of claim 226, further comprising:

sequencing DNA from the protein-producing cell; and

inserting at least a portion of the DNA in a host cell.

239. The method of claim 226, wherein the particle is fluorescent.

240. The method of claim 226, wherein the protein-binding agent is an antibody.

241. The method of claim 226, wherein the protein-binding agent is able to specifically bind the protein.

242. The method of claim 226, wherein the protein-producing cell is non-immortal.

243. The method of claim 226, wherein the protein-producing cell is a human cell.

244. A method, comprising:

removing blood cells from a subject;

encapsulating at least some of the blood cells in a plurality of aqueous microfluidic droplets contained within a fluid immiscible with the droplets, contained within a microfluidic channel, wherein the microfluidic droplets are substantially monodisperse and have an average diameter of less than about 1 mm;

at least partially separating, from the plurality of fluidic droplets, droplets containing antibody-producing cells from droplets that do not contain antibody-producing cells;

culturing the antibody-producing cells within the separated microfluidic droplets to secrete antibodies;

exposing the antibodies within the separated microfluidic droplets to a signaling entity comprising a microparticle and an agent, immobilized relative to the microparticle, able to bind the antibodies;

determining a characteristic of the antibodies by determining a change in a characteristic of the signaling entity in the presence of the antibodies contained within the microfluidic droplets; and

sorting the microfluidic droplets based on the determined characteristic of the antibodies produced by the antibody-producing blood cells.

245. A method, comprising:

encapsulating antibody-producing cells in a plurality of aqueous microfluidic droplets contained within a microfluidic channel, wherein the microfluidic droplets are substantially monodisperse and have an average diameter of less than about 1 mm;

culturing the antibody-producing cells within the microfluidic droplets to secrete antibodies;

exposing the secreted antibodies to a signaling entity contained within the microfluidic droplets, wherein the signaling entity comprises a microparticle and an agent, immobilized relative to the microparticle, able to bind the antibodies;

determining a characteristic of the antibodies by determining a change in a characteristic of the signaling entity in the presence of the antibodies contained within the microfluidic droplets; and

sorting the microfluidic droplets based on the determined characteristic of the antibodies produced by the antibody-producing blood cells.